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2-1994

New Morphological Characters for Identifying Individual Specimens of Haemonchus spp. (Nematoda: Trichostrongyloidea) and a Key to Species in Ruminants of North America

J. Ralph Lichtenfels Parasitic Disease Lab, ARS, United States Department of Agriculture, [email protected]

P. A. Pilitt United States Department of Agriculture, Agricultural Research Service

Eric P. Hoberg United States Department of Agriculture, Agricultural Research Service, [email protected]

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Lichtenfels, J. Ralph; Pilitt, P. A.; and Hoberg, Eric P., "New Morphological Characters for Identifying Individual Specimens of Haemonchus spp. (Nematoda: Trichostrongyloidea) and a Key to Species in Ruminants of North America" (1994). Faculty Publications from the Harold W. Manter Laboratory of Parasitology. 605. https://digitalcommons.unl.edu/parasitologyfacpubs/605

This Article is brought to you for free and open access by the Parasitology, Harold W. Manter Laboratory of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Faculty Publications from the Harold W. Manter Laboratory of Parasitology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. J. Parasitol., 80(1), 1994, p. 107-119 ? American Society of Parasitologists 1994

NEW MORPHOLOGICALCHARACTERS FOR IDENTIFYING INDIVIDUALSPECIMENS OF HAEMONCHUSSPP. (NEMATODA:TRICHOSTRONGYLOIDEA) AND A KEY TO SPECIES IN RUMINANTSOF NORTH AMERICA

J. R. Lichtenfels, P. A. Pilitt,and E. P. Hoberg BiosystematicParasitology Laboratory, Livestock and PoultrySciences Institute, U.S. Departmentof Agriculture,Beltsville, Maryland 20705-2350

ABSTRACr:The largestomach worms , Haemonchus placei, and Haemonchussimilis are importantpathogens ofcattle and sheep.This paperdescribes characteristics of surfacecuticular ridges (synlophe), which for the first time provide morphologicalcriteria for identifyingindividual adult specimens of either sex. The diagnosticpatterns of the synlopheon the anteriorhalf of specimenscan be observedat 400 x in temporary mounts on glass slides. The synlophecan be studiedin clearedpreserved specimens or in living or freshlythawed frozen specimens mounted in water. The synlophe of H. contortushas 30 ridges in the region of the posterior half of the esophagus,4 fewer than H. placei and H. similis. The 4 extra ridges of H. placei and H. similis are consistentlylocated bilaterallyto the 3 ventralmostand the 3 dorsalmostridges. The 4 extra ridgesof H. similis extend to the end of the synlopheposterior to midbody, but in H. placei they extend posteriorlyonly to the end of the anteriorquarter of the .A key is includedto the 3 species of Haemonchusparasitic in domestic sheep and cattle using characteristicsof spicules, female reproductivesystem, female tail, and the synlophe.

Three species of the genus Haemonchus Cobb, recognized features of the synlophe that can be 1898, are known to occur in domestic and wild used to determine the species of individual spec- ruminants of North America. They are common imens of either sex of H. contortus and H. placei. parasites of domesticated ruminants and have The third species of the genus Haemonchus achieved a worldwide distribution in part due to parasitic in North American ruminants is H. the movement of their economically important similis Travassos, 1914. This species has been hosts. These 3 species of large stomach worms reported in domestic cattle in a few southern are among the most pathogenic of states of the U.S.A., Central and South America, sheep, cattle, and goats worldwide, causing sig- Asia, and several Atlantic and Pacific islands nificant production losses due to morbidity, mor- (Donald, 1963), in Bos indicus in Tanzania and tality, costs of treatment, and suboptimal use of Uganda (Gibbons, 1979), domestic sheep in Bra- contaminated pastures (Gibbs and Herd, 1986). zil (Lins de Almeida, 1935), white-tail deer in Haemonchus contortus (Rudolphi, 1803) Cobb, Florida (Dinaburg, 1939), and in numerous other 1898, primarily a parasite of domestic sheep, ruminants and localities (Doss et al., 1985). In Ovis aries, also infects numerous other domestic addition to differences in the synlophe, distinc- and wild ruminants. tive characteristics of the spicules, female repro- Haemonchus placei (Place, 1893) Ransom, ductive tract, and female tail allow H. similis to 1911, is primarily a parasite of domestic cattle be separated from both H. contortus and H. placei. (Bos taurus) but it has been found also in do- An illustrated, dichotomous key to males and mestic sheep, white-tail deer (Odocoileus virgi- females of H. contortus, H. placel, and H. similis nianus), and pronghorn antelope (Antilocapra is presented. This key makes it possible to iden- americana). Haemonchus placei is not univer- tify individual specimens of all 3 species of large sally accepted as a separate species (Gibbons, stomach worms present in North American ru- 1979). However, Lichtenfels et al. (1986) de- minants. scribed differences between H. placei and H. con- Seven additional species of Haemonchus (re- tortus in the percentage of the body covered by view by Gibbons, 1979) that parasitize wild ru- the synlophe, which made it possible to identify minants of Africa and/or the camel (Dutt and most populations to species if at least 10 speci- Sahai, 1966) were not included in the present mens are examined. This paper describes newly study.

MATERIALSAND METHODS Received 2 April 1993; revised 10 September1993; A list of nematodes studied is presented (Table I) accepted 10 September 1993. includinghost names, geographiclocalities, numberof 107 108 THE JOURNALOF PARASITOLOGY,VOL. 80, NO. 1, FEBRUARY1994

TABLE I. Specimens of Haemonchus spp. studied by nematode species, host species, locality, and nematode sex.

Number studied Collection number* Species Host Locality Male Female

39960 Haemonchus contortus Ovis aries Florida 1 2 64756 H. contortus O. aries Georgia 0 1 70001 H. contortus O. aries Georgia 5 5 33156 H. contortus O. aries Maine 4 4 69990 H. contortus O. aries Maryland 1 2 70404t H. contortus O. aries Michigan 5 5 704051 H. contortus O. aries Michigan 5 5 70406? H. contortus O. aries Michigan 5 5 47825 H. contortus O. aries Nebraska 4 4 40172 H. contortus O. aries North Caroina 5 4 40659 H. contortus O. aries Texas 1 0 II H. contortus O. aries Texas 0 3 H. contortus O. aries Texas 1 1 H. contortus O. aries Texas 0 1 II H. contortus O. aries Texas 2 2 58830 H. contortus O. aries Vermont 0 5 56711 H. contortus O. aries West Virginia 3 1 69996 H. contortus O. aries Australia 2 1 81048 H. contortus O. aries Brazil 5 5 # H. contortus O. aries South Africa 12 10 70374 H. contortus Bos taurus Maryland 25 25 67080 H. contortus Capra hircus Sierra Leone 1 4 67185 H. contortus Odocoileus virginianus Oklahoma 2 2 66540? H. contortus O. virginianus Texas 6 0 32146** H. contortus Ovibos moschatus Washington, D.C. 5 2 70277 H. contortus Pelea capreolus South Africa 5 5 70276 H. contortus Redunca redunca South Africa 5 5 66649? H. contortus Syncerus caffer Uganda 3 3 39902 Haemonchus placei B. taurus Florida 2 1 69986 H. placei B. taurus Florida 5 0 70247 H. placei B. taurus Florida 3 2 49243 H. placei B. taurus Hawaii 0 5 69987 H. placei B. taurus Louisiana 5 5 70382 H. placei B. taurus Louisiana 16 10 47217 H. placei B. taurus Louisiana 4 6 58800 H. placei B. taurus Louisiana 3 0 29346 H. placei B. taurus Maryland 3 5 70324ff H. placei B. taurus Maryland 0 1 70372ff H. placei B. taurus Maryland 20 11 70373ff H. placei B. taurus Maryland 10 10 70375ff H. placei B. taurus Maryland 5 5 70377 H. placei B. taurus Maryland 6 6 70385ff H. placei B. taurus Maryland 0 1 19472 H. placei B. taurus Mississippi 4 5 70138 H. placei B. taurus New Jersey 2 1 H. placei B. taurus Texas 3 0 59090 H. placei B. taurus Guyana 3 3 70402 H. placei B. taurus South Africa 8 6 66540? H. placei O. virginianus Texas 1 2 H. placei O. aries Texas 0 1 69995 H. placei O. aries Australia 5 5 43625 Haemonchus similis O. virginianus Florida 1 0 34073 H. similis B. taurus Florida 0 4 39902 H. similis B. taurus Florida 9 4 70247 H. similis B. taurus Florida 1 2 70301ff H. similis B. taurus Maryland 7 5 70324ff H. similis B. taurus Maryland 8 2 70375ff H. similis B. taurus Maryland 0 2 70385ff H. similis B. taurus Maryland 20 1 45184 H. similis B. taurus North Carolina 0 3 29412 H. similis B. taurus Puerto Rico 2 2 29462 H. similis B. taurus Texas 1 2 59091 H. similis B. taurus Guyana 3 3 66648 H. similis Bos indicus Uganda 3 3

* Collection numbers of specimens deposited in the U.S. National Parasite Collection. t Susceptible strain from G. A. Conder, Kalamazoo, Michigan. LICHTENFELSET AL.-KEY TO SPECIESOF HAEMONCHUSIN NORTHAMERICA 109

LEGEND 1*mean * mean *--28E of mean LEGEND **....*28Eof mean SI " | |.- range : : range H, slmills H. placel Right barb J Spicule length ! I H. slmills H. contortus Left barb SSpicule length

H. 0ImIi0 H. placel H.placel Spicule length Left barb Right barb I' I'o I? I'• ? ,'I o ,,I 5 H. contortus H. contortus FIGURE 1. Spicule lengths (from data in Table II) Left barb Right barb of 3 species of Haemonchus with 95% confidence bands I i I I I I I i i for mean (?2 SE) and population (?2 SD). 10 20 30 40 50 60 70 80 90 FIGu1E 2. Distances (from data in Table II) from spicule barbs to distal tips of spicules of 3 species of specimens examined, and collection numbers of spec- Haemonchus with 95% confidence bands for mean (?2 imens deposited in the U.S. National Parasite Collec- SE) and population (?2 SD). See also Figures 10-12. tion (USDA, ARS, BARC-East, Bldg. No. 1180, Belts- ville, Maryland). Frozen specimens from T. M. Craig were used (Zarlenga et al., 1994) in DNA studies and shaped, but the pars ejectrix also includes a cylindrical therefore not deposited. Specimens from South Africa part that is clearly distinguishable from the thinner- loaned for study by J. A. van Wyk were returned to walled vestibule (Figs. 13-15). The infundibulum is him. equivalent to the pars haustrix of Veglia (1915). Whole specimens were cleared in phenol-alcohol (80 Populations of nematodes were identified to species parts melted phenol crystals and 20 parts absolute eth- as follows: H. similis were distinguished from H. con- anol) and studied in temporary mounts. Interference- tortus and H. placei on the basis of spicular structure contrast light microscopy was used to study the syn- and length, and vulvar structure and tail length. Then lophe and other characters at a magnification of 400 x. H. contortus and H. placei were separated on the basis Frozen specimens can be studied in water. Living spec- of spicule lengths (Lichtenfels et al., 1988) or on the imens should be studied in saline, and the coverglass percentage of the body bearing the longitudinal surface should be supported by glass beads if the viability of cuticular ridges of the synlophe (percentage of total the specimen is to be preserved. We have not tested the viability of such specimens. Cross sections were studied as either freehand cuts ....man made with a cataract knife and mounted in glycerine LEGEND ...*28E of mean jelly or in paraffin-embedded sections cut at 5 with , I /m a microtome and stained in hematoxylin and eosin. I,..I I Measurements of specimens were obtained with the H. place! tail aid of a calibrated ocular micrometer on a compound microscope and are recorded in micrometers unless •- I, ,1n indicated otherwise. Drawings of the synlophes were H. contortus free-hand while the at a / 9 tail prepared viewing specimens H. similis of 400 x. 3 06 magnification tail 00 0 The of the follows the detailed description ovejectors I I I I I I I description of the ovejectors of H. contortus by Veglia 0 100 200 300 400 500 600 700 800 (1915). The terminology of Veglia (1915) is modified as follows. The elongate cylindrical sphincters of Stron- FIGURE 3. Tail lengths (from data in Table III) of gyloidea (see Lichtenfels, 1980) are homologous to the females of 3 species of Haemonchus with 95% confi- pars ejectrix of Veglia (1915). The anterior portion of dence bands for mean (?2 SE) and population (?2 the pars ejectrix of Haemonchus spp. is sphincter- SD). See also Figures 16-18.

$ Ivermectin/benzimidazole-resistant strain from G. A. Conder, Kalamazoo, Michigan. ? Levamisole/benzimidazole-resistant strain from G. A. Conder, Kalamazoo, Michigan. IIAll specimens (6 lots) from T. M. Craig, Texas. (1) from Uvalde County, ivermectin/levamisole resistant; (2) original host Addax nasomaculata, with experimental infections in lambs; (3) from the Craig ranch; (4) from east Texas, original host Capra hircus with experimental infection in lambs, benzimidazole/ivermectin resistant; (5) from Brazos County, raised in cattle; (6) from Boerne, Kendall County. Sheep grazed on pasture with exotic antelope. # Specimens from sheep in South Africa from J. A. van Wyk. ? Possible hybrid specimens; short or intermittent subventral and subdorsal ridges. ** Specimens from a National Zoological Park animal. Original locality unknown. t"Experimental infections in calves in Maryland, originally from tracer calves in Florida. 110 THE JOURNALOF PARASITOLOGY,VOL. 80, NO. 1, FEBRUARY1994

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lengthin males and percentageof prevulvarlength in length), but the difference in range of measure- females) (Lichtenfelset al., 1986). Specimens previ- ments between H. similis and H. identifiedin the cited earlier studies were used placei (Fig. 2) ously is not bars when possible. The numbers of specimens listed in statistically significant (see overlapping Table I are those used to evaluatethe newly described for 2 SD). Thus, the barbs of the right spicules synlophecharacteristics. of H. placei and H. similis can be distinguished Samples were selected to representas wide a geo- by the percentage of total length from the distal graphicand host rangeas possible.Measurements were tip to the barb but not by actual measurements made of a few specimensfrom each of a largenumber of individual hosts because variation among samples because of the significantly greater spicule lengths from individual hosts was expectedto be greaterthan (Fig. 1) of the latter species. The means of the variationwithin one sample(Lichtenfels, unpubl. data). distances from spicule barb to distal tip can also Observeddifferences in size of variousstructures among be used to and H. contortus the 3 were separate H. placei species(Tables II, III) comparedstatistically but the of measure- by calculating95% confidencebands for the samples males, overlapping ranges (?2 SD) and for the sample means (?2 SE) (Steel and ments prevent the use of this character for iden- Torrie, 1960). The resultswere illustratedgraphically tification of individual males of these species. as described Lichtenfelset al. (Figs. 1-3) by (1988). Females of H. similis can be distinguished from those of H. placei their shorter RESULTS by significantly tail length (Figs. 3, 16-18; Table III); and most Size differences specimens ofH. similis can be distinguished from both H. placei and H. contortus by the presence Morphometrics of H. contortus, H. placei, and of a longer vagina (Table III) that usually extends similis are given by sex in Table II (males) H. into a vulval flap and opens in the flap (Fig. 15) and Table III (females). Among males of the 3 rather than on or near the body wall as in the species, the most useful size differences are spic- other 2 species (Figs. 13, 14). ule lengths (Fig. 1) and distances of the barbs from the distal end (Fig. 2). Figure 1 shows that the mean spicule lengths of all 3 species differ significantly from each other (95% confidence Synlophe level), but that overlapping ranges of spicule The synlophe in all 3 species is bilaterally and lengths prevent identification of individual spec- dorsoventrally symmetrical with the result that imens. The relative position of the distal barbs left and right, and also dorsal and ventral halves, of the spicules (Figs. 2, 10-12) of H. similis can are mirror images of each other. There is no ridge be used to distinguish unequivocally males of at the lateral line so each of the 30 or 34 ridges this species from those of H. contortus and H. is in either the dorsal or ventral field (Figs. 4-7). placei. Of the 3 species, only in H. similis are the In all 3 species the third ridge from the lateral barbs of both the left and right spicules greater chord both dorsally and ventrally is shorter than than 10% of the spicule length from the distal adjacent ridges and usually extends anteriorly tips. The barb of the left spicule of H. similis is only to the level of the cervical papillae, although significantly farther (20-21% of spicule length) in some specimens it ends well anterior to the from the distal tip than in the other 2 species excretory pore. (both about 11%). The barb of the right spicule Both H. similis and H. placei bear 17 dorsal of H. similis is significantly farther from the dis- and 17 ventral (total 34) longitudinal cuticular tal tip (13-17% of spicule length) than in H. con- ridges in the region of the posterior half of the tortus and H. placei (both about 5% of spicule esophagus (Figs. 5-7, 23, 27). In H. contortus,

FIGURES4-7. Diagrammatic drawings of the anterior ends of 3 species of Haemonchus showing the synlophe patterns in the region of the esophagus (dashed lines, sublateral ridges; dot-dash lines, subventral or subdorsal ridges; EI, esophageal-intestinal junction) (scale bar = 100 Lm).Note that in ventral views (Figs. 4-6) because there are no lateral ridges dividing ventral and dorsal fields, the lateralmost lines in these figures represent the lateralmost ridges of the dorsal field. 4. Anterior synlophe of H. contortus, ventral view, showing 17 of 30 ridges at EI. Note absence of subventral ridges. 5. Anterior synlophe of H. placei, ventral view, showing 19 of 34 ridges at EI. Note presence of subventral ridges. 6. Anterior synlophe of H. similis, ventral view, showing 19 of 34 ridges at EI. Note presence of subventral ridges. 7. Lateral view of H. similis showing one ridge of each pair of subdorsal and subventral ridges, which are present in H. similis and in H. placei. 112 THE JOURNALOF PARASITOLOGY,VOL. 80, NO. 1, FEBRUARY1994

FIGURES8-12. Lateral synlophe patterns and spicule tips of 3 species of Haemonchus (scale bars = 25 gm). 8. Lateral synlophe of H. contortus showing distal ends (arrows) of more lateral of the sublateral ridges ending next to paired lateralmost ridges. 9. Lateral synlophe of H. similis showing distal ends (vertical arrows) more dorsal and ventral of sublateral ridges adjacent to more lateral pair of sublateral ridges (horizontal arrows) adjacent to paired lateralmost ridges. 10. Spicule tips of H. contortus. 11. Spicule tips of H. placei. 12. Spicule tips of H. similis. LICHTENFELSET TO SPECIESOF HAEMONCHUSIN NORTHAMERICA 113 AL.-KEY

TABLEII. Morphometrics of Haemonchus spp. males.*

Haemonchus Haemonchus Haemonchus Character contortus placei similis

Number examined 23 21 24 Body length (mm) 11.0-17.0 (13.1) 10.9-18.9 (14.4) 7.3-10.1 (8.8)t Nerve ring1: 188-326 (254) 235-319 (287) 210-244 (228) Excretory pores 232-356 (285) 266-367 (321) 225-274 (246) Cervical papillaeg 271-462 (354) 351-440 (403) 281-353 (3!2) Subventral esophageal gland orifices1: 356-626 (493) 506-668 (587) 394-506 (448) Esophagus length (mm) 1.09-1.55 (1.26) 1.31-1.74 (1.48) 0.82-1.28 (1.10) Esophagus as percentage of body length 8.0-11.2 (9.8) 8.6-12.1 (10.4) 9.9-14.8 (12.6) Gubernaculum 195-255 210-270 142-198 length (221) (245) (179)f Spicule length 383-475 (425) 438-511 (481) 304-389 (341)f Spicule barb length right/left 37-48 (42)/19-24 (22) 45-60 (55)/22-32 (27) 62-81 (71)t/41-65 (54)f

* Measurements (in micrometers unless noted otherwise) are ranges followed by means in parentheses. Measurements that differ significantly (95% confidence) from those of at least 1 of the other 2 species. t $Measured from anterior extremity.

TABLEIII. Morphometrics of Haemonchus spp. females.*

Haemonchus Haemonchus Haemonchus Character contortus placei similis

Number measured 22 21 26 Body length (mm) 14.8-27.2 (18.5) 12.5-25.5 (19.6) 10.4-17.0 (13.4) Nerve ringt 210-394 (271)1: 225-330 (290) 165-300 (229) Excretory poret 205-400 (290) 281-379 (330) 188-289 (246) Cervical papillaet 243-484 (363) 359-460 (414) 233-379 (313) Subventral esophageal gland orificest 435-656 (525)1: 540-728 (623) 443-564 (500) Esophagus length (mm)t 1.15-1.66 (1.38) 1.38-1.84 (1.60) 1.09-1.42 (1.25) Esophagus as percentage of body length 5.8-8.9 (7.6) 6.7-11.1 (8.3) 7.8-11.5 (9.5) Vulva position (percentage)f 76.3-84.8 (82.4) 74-84 (81) 75.6-89.1 (81.7) Anterior infundibulum length 188-394 (272) 188-461 (290) 149-319 (204) Anterior spincter length 154-284 (222) 191-356 (244) 124-249 (184)l"** Vestibula length 150-263 (198) 225-356 (261) - Vagina length 75-188 (110) 75-131 (104)# 161-300 (217)?tt Posterior sphincter length 158-266 (227) 182-330 (245)1: 143-300 (216)11 Posterior infundibulum length 169-341 (277)? 240-441 (306) 152-300 (205)11 Tail length 251-530 (396) 378-720 (527) 135-278 (219)11 Phasmids (right/left)14 86-235 (143)/84-249 (143) 122-239 (167)/125-239 (171) 52-93 (74)?tt/60-93 (77)11

* Measurements (in micrometers unless noted otherwise) are ranges followed by means in parentheses. t Measured from anterior extremity. 1 n - 20. ? n =21. IIn = 22. #n= 19. ? n - 24. ** Measurement includes vestibula length. ft Measurements that differ significantly (95% confidence) from at least 1 of the other 2 species. $$Distance from distal tip of tail. there are only 15 ridges dorsally and 15 ventrally herein referred to as subventral ridges. There are (total 30) (Figs. 4, 19). This number was constant also 2 similar "subdorsal" ridges in an equivalent in both males and females in all lots that were position, subdorsally in these 2 species. The sub- studied, but minor variations were frequently ventral and subdorsal ridges usually extend an- observed due to either the presence of short extra teriorly only to a level equal to the middle third ridges in atypical locations (Figs. 6, 7) or cross- of the esophagus but more rarely as far anteriorly overs where one ridge reversed positions with an as the cervical papillae or excretory pore. They adjacent ridge. are continuous for most of their length but may Two of the 4 additional ridges present in H. be interrupted near their anterior and posterior similis and H. placei are illustrated as dot-dash ends. Haemonchus placei differs from H. similis lines in Figures 5 and 6. They are situated 1 on in that the subventral and subdorsal ridges of the each side of the 3 ventralmost ridges and are former extend posteriorly for 4-5 mm and end 114 THE JOURNALOF PARASITOLOGY,VOL. 80, NO. 1, FEBRUARY1994

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FIGURES13-18. Paired, opposed ovejectors and tails of females of 3 species of Haemonchus (i, infundibula; s, sphincters; v, vestibule; arrows, both ends of vagina and at junction of sphincters and vestibule) (scale bars = 100 gim). Note that variation in the anatomy of vulval lobes within species is high and is not a reliable character for identifying species. 13. Ovejectors of H. contortus with large lobe over vulva. 14. Ovejectors of H. LICHTENFELSET AL.-KEY TO SPECIESOF HAEMONCHUSIN NORTHAMERICA 115 near the middle of the length of the synlophe in males and 0.7-2.2 mm from the end of the (about one-fourth of the length of the nematode synlophe in females, reducing the number of from the anterior end), but in H. similis the sub- ridges to 22 (Fig. 26). In H. similis the number ventrals and subdorsals extend to the end of the of ridges is reduced by 4 twice: from 34 (Fig. 27) synlophe, which is near midbody. to 30 near the middle of the synlophe (3-4 mm The synlophe of experimentally produced hy- from the anterior end) (Figs. 28, 29) and then to brids (Lichtenfels et al., 1986) from matings of 26 (Fig. 30) 2-3 mm anterior to the end of the H. contortus and H. placei in sheep have an in- synlophe in females and near the end of the syn- termediate pattern of subventral ridges. When lophe in males. the 2 subventral and 2 subdorsal ridges were In both H. placei and H. contortus, the sub- present they were much shorter than in H. placei lateral ridges next to the lateral most ridges end and usually the subdorsals and subventrals were posteriorly first (Fig. 8), before the more dorsal absent on either the left or right side. and ventral of the sublaterals end, but the situ- A single lot of specimens identified as H. con- ation is reversed in H. similis (Fig. 9). tortus (No. 70404) had traces of subdorsal and to subventral ridges, but they were short (usually Key species less than 1 mm in length) and were not present The characters described above were used to in all 4 usual locations. construct an identification key to both males and In the anterior half of the body of all 3 species, females of the 3 species of Haemonchus parasitic 4 pairs of ridges (illustrated as dashed lines in in domesticated ruminants. Hybrids between H. Figs. 4-7) occur parallel to the lateralmost ridges contortus x H. placei have characteristics that (and are herein named the sublateral ridges). Pos- are intermediate between the 2 species (Lichten- teriorly in all 3 species the sublateral ridges dis- fels, unpubl. data). For a discussion of the use of appear at 2 different levels, 2 pairs (1 pair on the spicule lengths to identify species, see Lichtenfels left and 1 pair on the right) at about the middle et al. (1988). of the length of the synlophe and the second 2 la. M ale specimens 2 pairs more or less near the end of the synlophe...... lb. Female specimens 4 Because the pairs of sublateral ridges may not ...... 2a. Spicule length about 300-400 (Fig. 1) end posteriorly a completely symmetrical pat- /tm in and barbs of spicules near distal ends rela- tern (left pair may extend slightly longer than tively distant from distal tips (right 62-81, corresponding pair on the right, and/or ventral left 41-65; about 20 and 15% of spicule of pair may extend further than dorsal of pair) lengths, respectively) and right and left barbs and because the end becom- ridges frequently by in nearly symmetrical positions (Figs. 2, 12) ing discontinuous, it is difficult to find a perfectly Haemonchus similis .. symmetrical cross section showing the standard 2b. about 400-500 Spicule lengths /m (Fig. 1); number of sublateral ridges. In H. contortus the and barbs of spicules near distal ends rela- number of ridges is reduced from 30 to 26 (see tively close to tips with right barb about twice 25 ridges in Fig. 20) 3-4 mm from the anterior as far from as the left barb tip (37-60 /m) end and from 26 to 22 5-6 mm from the anterior 11 and 5% of (19-32 /Lm) (about spicule end and 2-3 mm anterior to the end of the syn- lengths, respectively) (Figs. 2, 10, 11) .. 3 lophe (Figs. 21, 22). Haemonchus placei loses the 3a. Mean spicule length of 10 specimens greater first 2 pairs of sublateral ridges 3-4 mm from the than 450 tm; or, synlophe with subventral anterior end, reducing the number from 34 (Fig. and subdorsal ridges (Fig. 5) that begin in 23) to 30 (Fig. 24); loses the 2 subdorsals and 2 region of esophagus and extend posteriorly subventrals 4-6 mm from the anterior end, re- 3-5 mm ...... Haemonchus placei ducing the number to 26 (see 25 ridges in Fig. 3b. Mean spicule length (of 10 or more random and loses the second 2 of sublaterals less than 450 25); pairs specimens) significantly /tm; 0.1-0.5 mm anterior to the end of the synlophe or synlophe without subventral or subdorsal

placei with vulva anterior to protruding knob. 15. Ovejectors of H. similis with small vestibule between sphincters and the long vagina extending to the vulva near the distal end of the long lobe. 16. Tail of H. contortusfemale. (Note: dorsal curvature of tail is of no significance.) 17. Tail of H. placei female. 18. Tail of H. similis female. 116 THE JOURNALOF PARASITOLOGY,VOL. 80, NO. 1, FEBRUARY1994

m.: -:iii,

25 29

26 3 22

26

FIGURES19-30. Cross sections of female specimens of 3 species of Haemonchus at 4 comparable body locations to show the number and structure of the ridges of the synlophe (arrows at lateral lines) (scale bars = 50 ptm). 19. H. contortus, at esophageal-intestinal (EI) junction, showing 30 ridges. 20. Haemonchus contortus, 4 mm from anterior end, showing 25 ridges. 21. Haemonchus contortus, 6 mm from anterior end, showing 22 ridges. 22. Haemonchus contortus, near midbody, showing 22 ridges. 23. Haemonchus placei, at EI, showing 34 ridges. 24. Haemonchus placei, 4 mm from anterior end, showing 30 ridges. 25. Haemonchus placei, 6 mm from LICHTENFELSET AL -KEY TO SPECIESOF HAEMONCHUSIN NORTHAMERICA 117

ridges (Fig. 4) or with only a trace of 1 or 2 Synlophe of them Haemonchus contortus ...... The distribution of surface cuticular ridges has 4a. without subventral or subdorsal Synlophe been used previously by Lichtenfels et al. (1986) or with a trace of 1 or 2 ridges (Fig. 4) only to distinguish populations of H. contortus from of them Haemonchus contortus ...... H. placei. The earlier studies (Lichtenfels et al., 4b. with subventral and subdorsal Synlophe 1986) described only differences in the propor- that extend from ridges (Figs. 5, 6) posteriorly tion of the body length bearing ridges to the un- the of the for several midlength esophagus ridged cuticle (the total body length in males and millimeters or more 5 ...... prevulvar length in females), but not in number 5a. Tail short and conical (135-278 Lm) (Fig. and pattern of distribution of ridges in the syn- vulva in vulval lobe distant from 18); usually lophe. Gibbons (1979), who did not recognize wall subventral and subdor- body (Fig. 15); H. placei, reported 32 ridges for H. similis and sal ridges extend to posterior end of syn- 22-30 for H. contortus but did not describe the Haemonchus similis lophe ...... pattern of distribution of the ridges. In the pres- Tail and almost filiform 5b. long (378-720 Lm) ent study we described 30-22 ridges on H. con- vulva flush with wall (Fig. 17); body (Fig. tortus, 34-22 on H. placei, and 34-26 on H. simi- subventral and subdorsal extend 14); ridges The more detailed description of the synlophe for about one-half the of lis. posteriorly length of H. contortus, H. placei, and H. similis reported the ...... Haemonchus synlophe placei herein was possible only because populations of H. placei and H. contortus could be identified DISCUSSION with confidence using the earlier characterization This report presents newly recognized char- of the synlophe of these species (Lichtenfels et acteristics of the 3 common large stomach worms al., 1986). of ruminants of North America that make it pos- The presence (in numerous populations from sible, for the first time, to identify individual a very wide geographic region, Table I) of a pair intact, whole adult specimens to species. The of subventral and a pair of subdorsal ridges in characters useful for identifying individuals can H. placei and H. similis and the absence of these be determined in fixed and cleared, living or ridges in H. contortus (with 1 exception in which freshly thawed frozen specimens at 400 x mag- traces of subdorsal and subventral ridges were nification. Previously only populations (not in- found) provided a useful character for identifying dividuals) of H. contortus and H. placei could be H. contortus. The principal value of this new identified by studying the anatomy of at least 10 synlophe character is in separating individual specimens to determine the percentage of the specimens of either sex of the similar species H. body bearing the synlophe (Lichtenfels et al., contortus and H. placei, but the difference in 1986), mean spicule lengths (Lichtenfels et al., length of the subventral and subdorsal ridges be- 1988), or the mean distance from the left spicule tween H. placei and H. similis is also useful as a barb to the distal tip (Roberts et al., 1954; Her- supplementary character for separating females lich et al., 1958). Previously described methods of these species. for identifying individual specimens include cy- In the present (Table I) as well as in previous tological methods (Bremner, 1955) to determine investigations, the synlophe characteristics were the presence or absence of a swollen chromo- found to be stable irrespective of the host species some in H. placei, and DNA polymerase chain (Lichtenfels, 1974, 1977; Lichtenfels and Pilitt, reaction primers to amplify variable DNA spacer 1983, 1991; Beveridge and Durette-Desset, 1992). regions within the ribosomal DNA repeat for Also, specimens of the Conder et al. (1990) strain which diagnostic bands can be visualized by aga- of H. contortus (No. 70404, Table I), which has rose gel electrophoresis (Zarlenga et al., 1993). traces of subventral and subdorsal ridges, had

anteriorend, showing 25 ridges. 26. Haemonchusplacel, slightly anteriorto midbody, showing 22 ridges. 27. Haemonchussimilis, at EI, showing 34 ridges. 28. Haemonchussimilis, 4 mm from anteriorend, showing 30 ridges. 29. Haemonchussimilis, 6 mm from anterior end, showing 30 ridges. 30. Haemonchus similis, near midbody, showing 26 ridges. 118 THE JOURNALOF PARASITOLOGY,VOL. 80, NO. 1, FEBRUARY1994 similar synlophe characteristics whether raised specimens) from individual hosts were relatively in lambs or gerbils, Meriones unguiculatus, al- homogeneous, and the means had small standard though the nematodes from gerbils were stunted errors, and were usually distributed above 450 (reduced in size and development of the repro- tm for H. placei and below 450 tm for H. con- ductive system). These results are consistent with tortus. Spicule length and barb position can be a previous test of the stability of synlophe char- used to identify H. similis. Thus, a study of the acteristics (Lichtenfels, 1974) in experimental and synlophe to determine the presence or absence naturally occurring populations of Nippostron- of subdorsal or subventral ridges is necessary only gylus braziliensis Travassos, 1914. From these to separate a few populations of H. placei or H. studies, it appears that host effects, even when contortus with unusual spicules such as in stunted causing severe stunting of growth and develop- specimens, specimens in mixed infections, or hy- ment of the nematode, do not affect the number brids; or, to confirm the identity of some female and pattern of distribution of the ridges of the H. contortus or H. placei. synlophe. The presence of subventral and subdorsal ridg- Female tail length es in H. placei and their absence in H. contortus was a consistent feature in all monospecific pop- The conical shape and short tail length of H. ulations studied (Table I) with one exception. similis females are especially useful for separat- The Conder strain of H. contortus from lambs ing the 2 species commonly found in cattle (H. (Coll. No. 70404, Table I) and gerbils (Conder placei and H. similis) in regions where they are et al., 1990) had a synlophe with short, asym- sympatric. In the U.S.A., H. similis has been metrical subventrals and subdorsals very similar found only in the southern states. The shape and to that found in experimentally produced hy- length of the female tail can be observed with brids. The possibility that the Conder strain low magnification and in combination with vul- (Conder et al., 1990) represents a hybrid was va position, living females of H. similis can be investigated. However, the reproductive vigor of identified in a dish with the aid of a dissection this strain (Conder et al., 1990) contrasts with microscope. Haemonchus contortus females have the extremely low reproductive potential of hy- tails of intermediate lengths between those of H. brids between H. contortus and H. placei re- similis and H. placei so the presence or absence ported by Le Jambre (1983a, 1983b). The Con- of subventral or subdorsal ridges must be deter- der strain was identified as H. contortus by a mined if the presence of H. contortus is suspect- DNA probe (Zarlenga et al., 1993) specific for ed. that species. This newly recognized character of the syn- lophe greatly enhances the feasibility of genetic Spicule morphology studies on these important pathogenic nema- The relatively long distance from the barbs to todes of sheep and cattle. Prior to this study only the distal tips of the spicules in H. similis, es- populations could be identified to species on the pecially in the left spicule, is an easily observed basis of the study of at least 10 specimens. Now, character for distinguishing this species from H. theoretically, individuals can be identified alive contortus and H. placei. Gibbons (1979) showed and then returned to a host and mated with an- that the relative positions of the spicule barbs other identified individual. With this new po- are quite variable among the species of the genus tential of identifying individual specimens to Haemonchus. Thus, because of the low vari- species, studies might be designed to shed some ability within species of this character and the light on many mysteries of Haemonchus of sheep large differences among species, it is a useful and cattle. For example, does one species do bet- character for identifying males to species, es- ter in some hosts or environmental conditions? pecially H. similis. How important are gene flow and selection pres- Lichtenfels et al. (1988) showed that spicule sure in maintaining the specific distinctness of lengths provided the quickest and easiest char- these closely related species under sympatric acter to use for separating most populations of conditions? In mixed grazing conditions what H. contortus and H. placei. Although there is happens to the nematode species composition, considerable overlap in the range of spicule population levels, and effects on the hosts? The lengths of H. contortus and H. placei, Lichtenfels prospects for research on these nematodes are et al. (1988) found that small samples (10-12 exciting indeed. LICHTENFELSET TO SPECIESOF HAEMONCHUSIN NORTHAMERICA 119 AL.-KEY

LITERATURECITED of ridges in the cuticle of Nippostrongylus brasi- liensis (Travassos, 1914) (Nematoda: Heligmo- BEVERIDGE,I., ANDM. C. DURETTE-DESSET.1992. The morphology of Nippostrongylus magnus, a para- somatoidea). Journal of Parasitology 60: 285-288. site of native Australian rodents. Transactions of *1977. Differences in cuticular ridges among spp. of North American ruminants with the Royal Society of South Australia 116: 109- 115. an illustrated key to species. Proceedings of the of 44:111- BREMNER,K. C. 1955. Cytological studies on the spe- Helminthological Society Washington cific distinctness of the ovine and bovine "strains" 119. . 1980. to of the of the nematode Haemonchus contortus (Rudol- Keys genera superfamily In CIH to the nematode phi) Cobb (Nematoda: ). Aus- Strongyloidea. keys par- asites of No. R. C. A. tralian Journal of Zoology 3: 312-323. vertebrates, 7, Anderson, G. and S. Willmott CONDER,G. A., L.-W. JEN, K. S. MARBURY,S. S. Chabaud, (eds.). Common- wealth JOHNSON,P. M. GUIMOND,E. M. THOMAS,AND Agricultural Bureaux, Farnham Royal, B. L. LEE. 1990. A novel anthelmintic model Bucks, England, 41 p. ANDP. A. PILITT. 1983. Cuticular utilizing jirds, Meriones unguiculatus, infected with -, ridge pat- of Nematodirus Haemonchus contortus. Journal of Parasitology 76: terns (Nematoda: Trichostrongy- 168-170. loidea) parasitic in domestic ruminants of North America with a to of DINABURG,A. G. 1939. Helminth parasites collected key species. Proceedings the of 50: 261- from deer, Odocoileus virginianus, in Florida. Pro- Helminthological Society Washington 274. ceedings of the Helminthological Society of Wash- AND . 1991. A of ington 6: 102-104. , redescription Oster- DONALD,A. D. 1963. The occurrence of Haemonchus tagia bisonis (Nematoda: Trichostrongyloidea) and a to of with a similis Travassos, 1914 in cattle in Fiji. key species Ostertaginae tapering Journal lateral from domestic ruminants in North of Helminthology 37: 179-184. synlophe America. of the Helminthological Society Doss, M. A., J. D. RAYBURN, J. H. SHAW,AND M. D. Journal KIRBY. 1985. Nematoda and nematode diseases. of Washington 58: 231-244. AND L. F. LE JAMBRE. 1986. Cuticular Supergenera, genera, species and subspecies: F-M. -, -, of Haemonchus contortus and Hae- Index-Catalogue of medical and zo- ridge patterns veterinary monchus ology. Special Publication No. 6, Part 3, Issued placei (Nematoda: Trichostrongyloidea). of the of July 1985, U.S. Department of Agriculture, Ag- Proceedings Helminthological Society Washington 53: 94-101. ricultural Research Service, U.S. Government AND . 1988. of Printing Office, Washington, D.C., 556 p. , Spicule lengths the ruminant stomach nematodes Haemonchus DUTT, S. C., AND B. N. SAHAI. 1966. Redescriptions contortus, Haemonchus and their of Haemonchus longistipes Railliet and Henry, placei, hybrids. of the of 1909 and bispinosus (Molin, 1860) with re- Proceedings Helminthological Society H. 55: 97-100. marks on the taxonomic status of placei Washington H. (Place, LINSDE J. 1935. Revisio do Hae- 1893) (Nematoda: Trichostrongylidae). ALMEIDA, genero Indian monchus 1898. Trichostron- Journal of Helminthology 18: 104-113. Cobb, (Nematoda: Memorias do Instituto Oswaldo Cruz 30: L. M. 1979. Revision of the genus Hae- gylidae). GIBBONS, 57-114. monchus Cobb, 1898 (Nematoda: Trichostron- F. H. H. N. AND M. MCKEVETT. gylidae). Systematic Parasitology 1: 3-24. ROBERTS, S., TURNER, 1954. On the distinctness of the ovine H. C., AND R. P. HERD. 1986. Nematodiasis specific GIBBS, and bovine "strains" of Haemonchus contortus in cattle: importance, species involved, immunity, Cobb and resistance. In The veterinary clinics of (Rudolphi) (Nematoda: Trichostrongyli- North Australian Journal of 2: 275-295. America. Parasites: Epidemiology and control, Vol. dae). Zoology R. G. AND J. H. TORRIE. 1960. 2, No. 2, H. C. Gibbs, R. P. Herd, and K. D. STEEL, D., Principles and of statistics. McGraw-Hill Book Murrell (eds.). W. B. Saunders Company, Phila- procedures New 481 delphia, p. 211-224. Co., York, p. F. 1915. The and of the HERLICH,H., D. A. PORTER,AND R. A. KNIGHT. 1958. VEGLIA, anatomy life-history Haemonchus contortus Third and Fourth A study of Haemonchus in cattle and sheep. Amer- (Rud). Annual of the Director of Re- ican Journalof VeterinaryResearch 19: 866-872. Reports Veterinary Union of South 349- LE JAMBRE,L. F. 1983a. Pre-mating barriers to spe- search, Africa, Pretoria, p. cies hybridization in Haemonchus. International 500. D. F. M. Journalfor Parasitology13: 365-370. ZARLENGA, S., STRINGFELLOW, NOBARY, AND J. R. LICHTENFELS.1994. and charac- 1983b. Pre-mating barriers in hybrid Hae- Cloning terization of ribosomalRNA fromthree monchus.? International Journal for Parasitology genes spe- 13: 371-375. cies of Haemonchus (Nematoda: Trichostrongy- LICHTENFELS,J. R. 1974. Number and distribution loidea). Experimental Parasitology (in press).